Nude mouse model for human neoplastic disease

ABSTRACT

A nude mouse model for human neoplastic diseases having histologically intact human neoplastic tissue transplanted onto an organ of the mouse which corresponds to the human organ from which the tissue is obtained.

RELATED APPLICATIONS

This application is a continuation of U.S. Ser. No. 08/169,735, filedDec. 17, 1993, now U.S. Pat. No. 5,491,284, which is a continuation ofU.S. Ser. No. 07/719,814 filed Jun. 24, 1991, now abandoned, which is acontinuation-in-part of U.S. Ser. No. 253,990 filed Oct. 5, 1988, nowabandoned, the contents of which are hereby incorporated by reference intheir entirety.

TECHNICAL FIELD

The present invention relates generally to a non-human mammalian modelfor human neoplastic diseases. More particularly, the invention relatesto a non-human mammalian model having neoplastic tissue, obtained from ahuman organ, transplanted to the corresponding organ of the model.

BACKGROUND

There has long been a need for a representative animal model for humanneoplastic disease. Such a model could serve many purposes. For example,it would be used to study the progression of neoplastic disease in humansubjects and assist in finding appropriate treatment. Such a model couldalso be used to test the efficacy of proposed anti-neoplastic agents.Additionally, an animal model could be employed in individualizedchemosensitivity testing of a cancer patient's tumors. The existence ofsuch a model would make drug screening, testing and evaluation much moreefficient and much less costly.

Some previous attempts at generating animal models for human neoplasticdisease employed transplantable animal tumors. There were tumors thathad developed in rodents and had been transplanted from animal toanimal, usually in inbred populations. Other animal tumor models weregenerated by inducing tumors in the animals by means of various agentsthat were carcinogenic, at least in the animal system. Still otheranimal tumor models were rodents containing spontaneously-occurringtumors. These rodent models, however, frequently responded tochemotherapeutic agents very differently than human subjects receivingthe same agent.

Another animal tumor model that developed starting some twenty years agoutilized mice without a thymus gland. These animals were deficient incellular immunity and had therefore lost their ability to reject foreigntransplant tissue. The mice, for reasons not clearly understood, wereessentially lacking in hair and came to be called “nude mice” or“athymic T-cell deficient nude mice.”

It was found that human tumors often grew when implanted subcutaneouslyunder the skin of nude mice, however, the take rate or frequency withwhich human tumor tissue actually formed a tumor in the mouse varieddepending on the individual donor and the tumor type. In these models,tumors that took exhibited histologically limited invasiveness andrarely metastasized, even if the original human tumor had been highlymetastatic. Accordingly, the subcutaneous nude mouse human tumor model,although better than the previously described rodent model, still hadsubstantial drawbacks, i.e. the subcutaneous transplants lacked theability to metastasize, and also were often more sensitive than thetumor in the patient in the original organ. The differences may be dueto the subcutaneous environment regarding pH, vascularity, accessibilityto drugs, etc.

Subsequent investigators found that invasion and metastases by humantumor cells in nude mice appeared to require that the cells be implantedorthotopically, i.e. injected into organs involved in the originalanatomical environment of the tumor. For example, Wang et al. (Exp. CellBiology, 50, 330 (1982)) report the expression of malignant phenotypewhen human colonic tumor cells were implanted by injection within thecolonic wall of nude mice. Moreover, Natio et al. (Cancer Research, 46,4109 (1986)) and Naito et al. (JNCI, 78,377 (1987)) report growth andmetastasis of tumor cells isolated from a human renal cell carcinoma andimplanted by injection into the kidneys of nude mice. More recently,Morikawa et al. (Cancer Research, 46, 6863 (1988) report the growth ofhuman colon carcinoma cells implanted by injection within the spleens ofnude mice.

While the human tumor model created by orthotopic implantation of humantumor cells in the nude mouse represents a significant advance overearlier models, the value of this model is clearly dependent on theextent to which the character of the original human tumor is maintainedin the immunodeficient host. Human tumor cells utilized in orthotopicimplantation are derived from tumor tissue that is disassociatedenzymatically. Enzymatic disassociation disrupts the architecture of thetumor tissue and thus the unique cellular organization. Cells behavevery differently when they are organized in a tissue structure asopposed to being disassociated.

Neoplasms are biologically heterogeneous, consisting of differentsubpopulations of cells having different biological behavior anddifferent metastatic potential (see Naito et al., Cancer Research, 46,4109-4115 (1986); Naito et al., JNCI, 78,377 (1987); and Morikawa etal., Cancer Research, 48, 6863 {1988}). Enzymatic disassociation oftumor tissue, the conventional method used to isolate tumor cells fromfresh surgical specimen, disrupts the original tumor architecture andprecludes obtaining a truly representative tumor cell population forimplantation. Enzymatic disassociation also alters cellular behavior anddrug response.

For example, in routine location of tumor cells for implantation orsensitivity testing, tumor tissue from a surgical specimen isdisassociated enzymatically to produce cells which are then implantedsubcutaneously (s.c.) in nude mice. The purpose of the s.c. implant isto produce a larger amount of tumor tissue for studies of predictivesensitivity for therapeutic agents as well as for implantation. Aftersufficient s.c. tumor growth occurs, the tumor is excised anddisassociated enzymatically. As mentioned previously, enzymaticdisassociation of the tumor cells disrupts the tumor architecture andconsequently cells that are selected for sensitivity testing ororthopedic implantation by injection may not be representative orcharacteristic of the original patient tumor.

Thus the art is presently lacking a truly adequate non-human mammalianmodel for human neoplastic disease. In particular, what is needed in theart is a model which has the ability to accurately mimic the progressionof neoplastic disease as it occurs in a human subject. Such models andmethods of generating same are disclosed and claimed herein.

SUMMARY AND OBJECTS OF THE INVENTION

The present invention relates to an improved non-human mammalian modelfor human neoplastic disease.

In a first aspect, the present invention provides a novel non-humanmammalian model for human neoplastic disease wherein histologicallyintact human neoplastic tissue is transplanted onto the correspondingorgan of the model, said model being sufficiently immunodeficient toallow the transplanted tissue to grow and mimic the progression ofneoplastic disease in the human donor.

In another aspect, the present invention provides a novel non-humanmammalian model for human neoplastic disease wherein neoplastic tissuefrom a human organ is implanted in a vascularized matrix created on thecorresponding organ of the immunodeficient model.

In a further aspect, the present invention provides a novel non-humanmammalian model for human neoplastic disease wherein human neoplastictissue is transplanted to the immunodeficient model by sandwiching theneoplastic tissue between an abdominal skin flap of the model and thecorresponding organ of the model.

In yet another aspect, the present invention provides a novel non-humanmammalian model for human neoplastic disease wherein neoplastic tissuefrom a human organ is transplanted to the immunodeficient model bysecuring, to the surface of the corresponding organ of the model, atleast two pieces of neoplastic tissue in close proximity to each other.

In still another aspect, the invention provides a method of generating anon-human mammalian model for human neoplastic disease, the methodcomprising, providing a laboratory animal having sufficientimmunodeficiency to allow implanted human neoplastic tissue to grow andmimic the progression of human neoplastic disease in the donor; bytransplanting neoplastic tissue from a human organ into thecorresponding organ of the immunodeficient animal.

In yet another aspect, the invention provides a method of generating anon-human mammalian model for human neoplastic disease, the methodcomprising, providing a laboratory animal having sufficientimmunodeficiency to allow implanted human neoplastic tissue to grow andmimic the progression of neoplastic disease in the human donor, securinga vascularizing matrix to a selected organ of the animal and allowingthe matrix to vascularize; and implanting neoplastic tissue from a humanorgan in the vascularized matrix wherein the matrix is located in thecorresponding organ of the model.

In still another aspect, the invention provides a method of generating anon-human mammalian model for human neoplastic disease, the methodcomprising, providing a non-human mammalian laboratory animal havingsufficient immunodeficiency to allow implanted human neoplastic tissueto grow and mimic the progression of neoplastic disease in the humandonor; and sandwiching neoplastic tissue from a human organ between anabdominal skin flap created in the model and the Corresponding organ ofthe model.

In yet a further aspect, the invention provides a method of generating anon-human mammalian model from human neoplastic disease, the methodcomprising, providing a non-human mammalian laboratory animal havingsufficient immunodeficiency to allow implanted human neoplastic tissueto grow and mimic the progression of neoplastic disease in the humandonor; and securing at least two pieces of neoplastic tissue from ahuman organ to the surface of the corresponding organ of the model.

DETAILED DESCRIPTION OF THE INVENTION

Copending parent application, U.S. Ser. No. 253,990 filed Oct. 5, 1988,discloses animal models for human neoplastic disease wherein humanneoplastic tissue is implanted into the corresponding organ of animmunodeficient animal that has sufficient immunodeficiency to allow thetransplanted neoplastic tissue to grow and mimic the progression ofneoplastic disease in the human donor. The method used to generate theanimal models disclosed in U.S. Ser. No. 253,990 is described in thefollowing paragraphs and in Examples I, II and III.

Animals that are suitable as immunodeficient hosts includes athymicrodents, i.e. rats and mice having no T-cell immunity. Particularlypreferred animals are athymic mice which are readily available and maybe obtained commercially from Charles River Laboratories, Inc.,Wilmington, Mass. (Catalog identification: Crl:nu/nu(CD-1)BR, homozygous28-42 days old).

The placement of neoplastic tissue in the immunodeficient host animalaccording to copending parent application, U.S. Ser. No. 253,990, iscarried out by means of orthotopic implantation. This refers to animplant or graft transferred to a position formerly occupied by tissueof the same kind. The terminology orthotopic implantation is used torefer to the grafting of histologically intact human neoplastic tumortissue from a human organ into the corresponding organ of animmunodeficient animal. Human neoplastic tissue that is utilizedcomprises tissue from fresh surgical specimens which are pathologicallydiagnosed tumors occurring in, for example, human kidney, liver,stomach, pancreas, colon, breast, prostate, lung, testis and brain. Suchtumors include carcinomas as well as sarcomas and implantation thereofencompasses all stages, grades and types of tumors.

Prior to implantation, the human neoplastic tissue is maintained byplacing it in a suitable nutrient medium, such as Eagle's MinimumEssential Medium containing ten percent fetal calf serum and a suitableantibiotic, such as gentamycin. The medium containing the tissue is thencooled to approximately 4° C. Tissue can be maintained in this mannerfor approximately twenty-four to seventy-two hours.

A selected tissue specimen is prepared for implantation by forming intoa mass a suitable size for insertion into a suitably prepared cavity inthe selected organ. The specimen size may vary from about 0.1×0.1×0.1 cmto about 0.2×0.1×0.1 cm. The technique used to form a specimen ofsuitable size comprises testing the tissue to size by pulling intopieces of the desired size with forceps or the like.

Microsurgical instruments typically used to carry out tissueimplantation include a castrovijeo needle holder, jeweler's forceps(straight and curved), iris forceps, iris scissors and straight andcurved tissue forceps, including one each with teeth and one eachwithout teeth.

Prior to implantation of neoplastic tissue, the selected immunodeficientanimal is anesthetized with a suitable anesthetic. Implantation of allorgan tissue, except lung tissue, is conveniently accomplished byconventional anesthesia using ethyl ether. When lung tissue isimplanted, pentabarbitol is used as the anesthetic.

Implantation of tissue from a hepatoma or tumor from a human liver iscarried out utilizing the caudal lobe of the recipient animal's liver asthe implantation site. Several loose sutures are placed over the lobeand an incision is made longitudinally under the liver serosa toaccommodate a tumor mass of approximately 0.1×0.1×0.1 cm in size. Afterplacement of the tumor mass in the incision, the sutures are pulledsnugly over the tumor in order to secure it in place.

The process of implantation of tissue from a human pancreatic tumor iscarried out by making an incision in the recipient animal's pancreas atthe head of the organ near the duodenum. Care is exercised to select ana vascular area. An incision is made in the selected area and a tumormass of approximately 0.1×0.1×0.1 cm is implanted in a manner identicalto that described in the preceding paragraph. Tissue from all stages andall grades of pancreatic carcinoma may be implanted in this manner.

The implantation of tissue from a human mammary carcinoma is carried outby surgically implanting the tumor in the mammary fat pad of a recipientfemale animal. The tumor mass is approximately 0.1×0.1×0.1 cm in size.After placement of the tumor in the pocket, the pocket is closed with asuture. All stages and grades of mammary carcinoma may be implanted inthis manner.

Implantation of tissue from a human prostatic carcinoma into theprostate of a recipient animal is carried out by surgically forming anopening in the prostate and then placing 5 tissue specimens ofapproximately 0.1×0.1×0.1 cm in size under the prostate capsule. Afterplacement of the tissue specimen, the opening in the capsule is closedwith appropriate sutures.

Implantation of tissue from a human testicular carcinoma into the testisof a recipient animal is carried out by penetrating the testis along thelongitudinal axis with a number-18 gauge needle and injecting a tumormass of approximately 0.1×0.1×0.1 cm in size through the needle. Whenthe end of the tumor specimen is visible at the tip of the needle, theneedle is gently withdrawn while visible tumor tissue is held in placewith forceps. The holes made by the needle is then closed with a singlesuture.

In preparation for implantation of neoplastic lung tissue into the lungsof the recipient animal, a tracheotomy is performed and plastic tubingis incubated. Thereafter, implantation may be effected by severalprocedures. In one implantation procedure, tracheotomy tubing isadvanced to reach either lung lobe(s); a small (0.1×0.1×0.1 cm) tumormass is injected through the tubing; and the tubing is then removed andthe tracheal wound is closed with a suture.

In the other implantation procedure, precautionary tubing is insertedinto the trachea; a small stab wound is made on the right chest to bringup a lobe of the right lung which plugs the thoracic cavity therebypreventing collapse of the lung; the lung lobe is gently clamped at thebase and two ligatures are loosely placed on the lung; an incision ismade on the lung, a tumor mass of approximately 0.1×0.1×0.1 cm isimbedded therein; the ligatures are snugly tied; and the lung lobe isplaced back into the thoracic cavity and the wound is closed.Tissue-from all stages and grades of small cell and non-small cell lungcarcinomas may be implanted by either of the foregoing procedures.

In order to implant neoplastic human brain tissue into the recipientanimal's brain, a bur hole is made through the parietal cranial bone ofthe animal. A tumor mass of approximately 0.1×0.1×0.1 cm is selected andimplanted in the brain. The hole in the cranial bone is then sealed bymeans of bone wax.

The present invention is an extension and improvement of the inventiondisclosed in copending parent application U.S. Ser. No. 253,990 filedOct. 5, 1988. In the present invention, a non-human mammalian model forhuman neoplastic disease is generated by improved methods oftransplanting histologically intact neoplastic tissue from a human organto the corresponding organ of an immunodeficient model that hassufficient immunodeficiency to allow the transplanted tissue to grow andmimic the progression of neoplastic disease in the human donor. Themethods used to generate the animal models of the present invention aredescribed in the following paragraphs and in Examples IV-VII.

Transplantation of neoplastic tissue from a human organ to thecorresponding organ of an immunodeficient animal as taught in thepresent invention is referred to as orthotopic transplantation. In thepresent invention, the terminology orthotopic transplantation is used torefer to the grafting of histologically intact human neoplastic tumortissue from a human organ onto the corresponding organ of animmunodeficient animal.

Human neoplastic tissue can be utilized in the present invention as wellas preparation of such tissue has been described earlier in accordancewith copending parent application U.S. Ser. No. 253,990.

One preferred method for transplanting human neoplastic tissue to animmunodeficient animal model according to the present invention utilizesa vascularizing matrix. The purpose of the matrix is to induce thedevelopment of blood vessels and thereby enhance the survival and growthof the transplanted neoplastic tissue. In this method, the matrix istransplanted on the appropriate organ by means of a surgical suture(s).When the matrix becomes well vascularized, which usually occurs in abouttwenty (20) days, the histologically intact specimen of human neoplastictissue is implanted directly into the vascularized matrix. The termvascularizing matrix as used herein refers to liquid-permeable,water-insoluble material having the general physical characteristics ofa sponge and being substantially absorbable in a living mammalian body.Specific examples of such materials are absorbable gelatin sponge andcellulose sponge. While absorbable gelatin sponge is the preferredvascularizing matrix, those skilled in the art will realize that anumber of materials can be utilized as the vascularizing matrix.

Another preferred method of transplanting human neoplastic tissueaccording to the present invention utilizes an internal skin flap overthe transplanted surgical specimen. Use of the skin flap inducesvascularization and take of the transplanted tissue. In this method, a Ushaped incision is made in the abdomen of the immunodeficient animalmodel and the resulting skin flap is lifted up and the abdominal wall isopened along the line alba. The cecum (or other organ) is accessedthrough the abdominal incision, and neoplastic tissue is placed betweenthe cecum serosa (or other organ) and the skin flap. Surgical suturesare applied along the edge of the skin flap to fix the flap to the cecum(or other organ). The cecum (or other organ) together with the skin flapis put back into the abdominal cavity and peritoneum and rectus musclesare closed with sutures. Finally, the skin layer is also closed withsutures and surgical adhesive is applied to ensure a good closure of theabdominal wall.

Still another preferred method of transplanting human neoplastic tissueto an immunodeficient animal model according to the present inventionutilizes multiple pieces of tissue arranged in a shish-kabobconfiguration. In this method, a thread-like material is passed throughat least two pieces of human neoplastic tissue and the resulting tissuearrangement is positioned on the surface of the corresponding organ ofthe immunodeficient mode. The shish-kabob configuration is attached tothe animal organ by securing a pair of terminal ends of the thread-likematerial to the organ. The term thread-like material as used hereinrefers to absorbable surgical suture such as, for example, Chromic Gutsurgical suture and Coated VicrylR surgical suture, both obtainable fromEthicon, Inc. located in Somerville, N.J. A particularly preferredvariation of this method of transplantation comprises interspersingpieces of normal tissue between pieces of neoplastic tissue in theshish-kabob configuration.

The animal models of the present invention are particularly useful instudying the progression of human neoplastic disease. These studies, incombination with other clinical testing modalities such as diagnosticimaging, help in the selection of the most appropriate form oftreatment.

For example, when an animal model of the present invention is subjectedto tumor imaging, the clinician is allowed to identify both primary andsecondary sites of tumor growth and to estimate the overall burden ofthe tumor on the animal. Tumor imaging is conventionally carried out byinjecting the animal model with a labeled anti-tumor antibody such as anantibody labeled with a radioactive isotope; allowing the antibody timeto localize within the tumor; and then scanning the animal using aradiation detector. When a computer is used to compile an image of theradioactivity detected in the animal's body, the computer can color codethe image according to the intensity of the radiation. Zones of highradioactivity in regions of the body not expected to accumulate theantibody or its metabolites indicate the possible present invention.

The animal models of the present invention can also be used to screennew anti-neoplastic agents to determine the ability to such agents toaffect tumors at the primary site and also at distant metastatic sitesor to prevent distant metastases from occurring. The models will be alsouseful for individualized chemosensitivity testing of a cancer patient'stumors.

Additionally, the animal models of the present invention are useful instudying the effects of nutrition on the progression of human neoplasticdisease. These studies can be particularly significant in view of thedemonstrated impact of various deficiencies on healthy subjects.

Examples I-III illustrate the invention which is set forth in copendingapplication U.S. Ser. No. 253,990, filed Oct. 5, 1988. Examples IV-X areprovided in order to illustrate the present invention and are not beconstrued as limiting the scope of the invention or as being inclusiveof all embodiments of the invention.

EXAMPLE I

In this example, fresh surgical specimens of tissue from a tumor excisedfrom a human kidney were transplanted into the kidneys of nude mice. Thetissue specimens, which were pathologically diagnosed as renal cellcarcinoma, were prepared to size by the teasing procedure describedearlier.

Five athymic nude mice age four (4) to six (6) weeks were selected asthe animal recipients for the implants. In preparation for surgery, themice were anesthetized with ether. An incision was made in each animalto access the kidney under the capsule. A wedge shaped cavity was formedby excision of the renal cortex of each recipient kidney and a mass oftumor tissue of approximately 0.1×0.1×0.1 cm was placed under the renalcapsule. A suture was then employed to secure the implant in place.

The five mice of this example were still alive six months later.Approximately one month following implantation of the tissue, the micewere surgically opened and the implanted tumors were observed. In eachcase, the tumor was found to have taken, i.e. the implanted neoplastictissue had invaded adjacent tissue. Histological analysis was performedon the tissue implants at this time. Such analysis comprised removingtissue samples from each animal and comparing the samples with a tissuesample from the tissue donor.

Preparation of the tissue samples for histological analysis was carriedout by (1) fixing the sample in formalin; (2) embedding the fixed samplein paraffin; (3) preparing 5-micron sections of the fixed, embeddedsample; (4) staining the sections with hematoxylin and cosin; and (5)microscopically observing the tissue structure in each section.

Histological analysis revealed that the tissue in the recipient animalspreserved its architecture and tissue type and mimicked progression ofthe disease in the human donor.

EXAMPLE II

In this example, specimens of human tissue excised from the stomach andpathologically diagnosed as gastric carcinoma were prepared to size bythe teasing procedure described earlier.

Five athymic nude mice age four (4) to six (6) weeks were selected asthe animal recipients for the implants. In preparation for surgery, themice were anesthetized with ether.

Each anesthetized mouse was opened to provide access to the stomach. Anincision was made in the stomach wall using a number 11 scalpel takingcare to penetrate the mucosal layer. A pocket was formed large enough toreceive five tumor masses of about 0.1×0.1×0.1 cm each. A tumor piece ofapproximately this size was selected and inserted into the pocket andthe incision was cloned using a 7-0 suture.

The five mice of this example have survived for about three (3) to four(4) months and otherwise appear healthy. Subsequent surgical opening ofthe stomach of these mice has verified that the tumors have taken.

EXAMPLE III

In this example, specimens of human tissue removed from a human colonand pathologically diagnosed as colon carcinoma were prepared to size bythe testing procedure described earlier.

Five athymic nude mice, age four (4) to six (6) weeks were selected asthe animal recipients for the implants. In preparation for surgery, themice were anesthetized with ether. Each anesthetized mouse was opened toprovide access to the colon. A pocket or cavity was surgically formed inthe seromuscular layer with care exercised not to enter the lumen. Fiveto ten tumor masses of approximately 0.1×0.1×0.1 cm each were insertedinto the pocket which was then closed with a suture.

Four of the five mice which underwent this implant surgery have survivedfor three to four months and appear to be in a good health.Approximately one month following tissue implantation, the mice weresurgically opened and the tumors were observed to have taken.

EXAMPLE IV

This example relates to the use of a vascularizing matrix to inducevascularization and take of orthotopically transplanted human tumortissue.

A surgical tissue specimen, removed from a human colon andpathologically diagnosed as colon carcinoma, was washed with colon-washmedium. Necrotic tissue was removed and the tumor was then cut intosmall pieces (about 1-mm³). Colon-wash medium, used to remove infectiousintestinal material, was formulated by combining 500 ml of MinimumEssential Medium with Earle's salts (MEM Earle's) with 70 ml fetalbovine serum, 75.2 mg Penicillin G sodium salt, 125 mg Streptomycin, 10ml Fungizone antibiotic (250 ug amphotericin B and 205 ug sodiumdeoxycholate/ml in deionized distilled water), 5 mg Tetracycline, 50 mgAmikacin, 75 mg chloramphenicol and 50 mg Gentamycin.

GelfoamR brand of absorbable, sterile gelatin sponge (obtained from TheUpjohn Co., Kalamazoo, Mich.) was hydrated with MEM Earle's. Thehydrated sponge was cut into approximately 0.3-0.5×0.3-0.5×0.3-0.5 cmpieces which were transplanted onto the cecum of nude mice by means of asimple surgical suture on top of the cecum serosa. After 20 days, thesponges became well vascularized.

The transplanted vascularized sponges were cut in the center to make apocket and about 10-15 of the previously prepared 1-mm³ tumor pieceswere implanted into each pocket which was closed by means of a surgicalsuture. The tumor grew locally and regional as well as liver metastasesoccurred.

EXAMPLE V

This example relates to the use of an internal skin flap to inducevascularization and take of orthotopically implanted human tumor tissue.

The tumor tissue used is identical to tissue used in Example IV and wasprepared for implantation according to the procedure described inExample IV.

Skin flaps were constructed in the lower abdomen of nude mice by makingincisions along three sides of a rectangular area (a U-shaped incision).The flap was lifted up and the abdominal wall was opened along the linealba. The cecum was exteriorized from the abdominal cavity and tumorpieces (about 1-mm³) were placed between the cecum serosa and the skinflap. Surgical sutures were applied along the two opposing edges of theflap to fix the flap on the cecum. The cecum, together with the skinflap was put back into the abdominal cavity. Peritonerum and rectusmuscles were closed with sutures followed by reattachment of the skinflap with sutures. As a last step, surgical adhesive was applied toensure good closure of the abdominal wall. The tumor grew at theimplanted site and formed abdominal metastases.

EXAMPLE VI

This example relates to the use of a shish-kabob tissue configuration toeffect orthotopic transplantation of human tumor tissue.

A surgical tissue specimen, removed from a human colon andpathologically diagnosed as colon carcinoma, was well washed withcolon-wash medium. Necrotic tissue was removed and the tumor was thencut into small 1-mm³ pieces. Eight of the 1-mm³ pieces were assembled ina shish-kabob configuration by stringing the pieces together on a pieceof surgical suture.

The shish-kabob tissue configuration was transplanted in nude mice byplacement of the configuration on the mouse colon approximately 0.5 to 1cm away from (i.e., up-stream of) the animals cecum. The configurationis held in place by securing the terminal ends of the suture material tothe organ of the animal.

EXAMPLE VII

As shown below, local growth, regional metastasis, and in someinstances, distant organ metastasis have been achieved using the threenovel methods of orthotopic transplantation described in the presentinvention.

TABLE 1 Orthotopic Transplantation of Human Colon Carcinoma to the Colonof Nude Mice Number of Local Regional Distant Method Animals GrowthMetastasis Metastasis Matrix 3 56-62 days 56-62 days 160 days Skin Flap3 48-62 days 48-62 days Shish- 4 62-138 days 62-138 days kabob

EXAMPLE VIII

This example relates to the use of surgical adhesive to glue a piece oftumor tissue onto the top of the urinary bladder of the nude mouse.

A piece of tumor tissue specimen about 2-mm³ size was prepared for tumortransplantation. The nude mouse was operated on under surgicalanesthesia with the full exposure of the urinary bladder. A small amountof surgical adhesive (2-cyanoacrylic acid ester) was applied on the topof the urinary bladder and the previously prepared tumor piece was thenglued onto the top of the urinary bladder. The abdomen was closed withsurgical sutures.

Using the transplantation method described above, we transplanted theras-transfected human bladder RT-10 carcinoma cell line xenograft. As aresult, we achieved unexpected extensive growth and metastases,including invasion of the whole thickness of the urinary bladder wall,lymph node metastases, and multi-organ metastases in the liver,pancreas, spleen, ovary, kidney, ureter and lung. This transplantationresult of RT-10 is in striking contrast to the result obtained whenRT-10 was injected transurethrally as disaggregated cells where onlylocal invasion and no distant metastasis were observed. (Theodorescu etal., Proc. Natl. Acad. Sci(1990), Vol 87, 9047-9051.)

EXAMPLE IX

In this example, a human tongue cancer specimen, which was prepared bybeing cut into 1-mm³ pieces, was transplanted orthotopically to thefloor of the mouth of a nude mouse.

An incision was made along the midline on the upper neck of the mouse.After blunt dissection of the muscles of the floor of the mouth, fivepieces of prepared tumor tissue were implanted in between the musclesdeep in the floor of the mouth. Surgical sutures were applied to closethe dissected muscles and skin layer.

Extremely invasive growth was observed which involved the whole jaw aswell as deep in the nasopharynx. This is distinctly different formgrowth observed when tumor pieces were implanted subcutaneously in theneck area. The subcutaneously grown tumor was completely encapsulatedand exhibited no invasion of the adjacent tissue.

EXAMPLE X

In this example, a human pancreatic tumor specimen was prepared by beingcut into 1-mm³ pieces, and tumor pieces were transplanted onto the nudemouse pancreas.

A midline incision was made on the upper abdomen of the nude mousemuscle layers and the peritoneum were opened along the line alba. Ten(10) pieces of previously prepared tumor pieces were assembled in ashish-kebab configuration by striking the pieces together on a piece ofsurgical suture. The configuration was secured on the pancreas. Theabdomen was then closed with one layer surgical suture.

Two different kinds of human pancreatic cell line xenografts weretransplanted as described above. Invasive growth was observed into bothcases, including the invasion of the duodenum and spleen.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be obvious that certain changes and modificationsmay be practiced within the scope of the appended claims.

1. A nude mouse model for progression of human neoplastic disease, theprogression of said disease being characterized by growth of a primarytumor site and metastasis to secondary tumor sites, wherein said mouseis characterized by having has histologically intact human neoplastictissue of at least 1 mm³ in size transplanted onto an organ of saidmouse which corresponds to the human organ from which said tissue isoriginally obtained; and havinghas sufficient immuno-deficiency to allowsaid transplanted neoplastic tissue to grow andat said primary site andmetastasize to said secondary tumor sites, so as to mimic theprogression of the neoplastic disease including the metastatic behaviorof said neoplastic disease in the human donorhumans.
 2. A nude mousemodel according to claim 1 wherein said neoplastic tissue is selectedfrom breast tissue, ovarian tissue or and pleural tissue.
 3. A nudemouse model according to claim 2 wherein said neoplastic tissue isobtained from human breast tissue.
 4. A nude mouse model according toclaim 3 wherein said human neoplastic breast tissue is implanted in themammary fat pad of the mouse.
 5. A nude mouse model according to claim 2wherein said neoplastic tissue is obtained from human ovarian tissue. 6.A nude mouse model according to claim 5 wherein said human neoplasticovarian tissue is implanted in the ovarian capsule of the mouse.
 7. Anude mouse model according to claim 5 wherein said human neoplasticovarian tissue is transplanted by securing to the surface of the mouseovary at least two pieces of neoplastic tissue in close proximity toeach other.
 8. A nude mouse model according to claim 2 wherein saidneoplastic tissue is obtained from human pleural tissue.
 9. A nude mousemodel according to claim 8 wherein said neoplastic tissue is implantedin the parietal pleura of the mouse.
 10. A nude mouse model according toclaim 9 wherein said neoplastic tissue is implanted in the visceralpleura of the mouse.
 11. A method of generating a nude mouse model forprogression of human neoplastic disease, the progression of said diseasebeing characterized by growth of a primary tumor site and metastasis tosecondary tumor sites, said method comprising: transplantinghistologically intact human neoplastic tissue of at least 1 mm³ in sizetransplanted onto an organ of a nude mouse which corresponds to thehuman organ from which said tissue is originally obtained; and allowingsaid transplanted tissue to grow and at said primary site andmetastasize to said secondary tumor sites, so as to mimic progression ofthe neoplastic disease including the metastatic behavior of saidneoplastic disease in the human donor humans.
 12. A method of generatinga nude mouse according to claim 11 wherein said human neoplastic tissueis selected from breast tissue, ovarian tissue or and pleural tissue.13. A nude rodent model for progression of human neoplastic disease, theprogression of said disease being characterized by growth of a primarytumor site and metastasis to secondary tumor sites, wherein said rodenthas histologically intact human neoplastic tissue of at least 1 mm³ insize transplanted onto an organ of said rodent which corresponds to thehuman organ from which said tissue is originally obtained; and hassufficient immuno-deficiency to allow said transplanted neoplastictissue to grow at said primary site and metastasize to said secondarytumor sites, so as to mimic the progression of the neoplastic diseaseincluding the metastatic behavior of said neoplastic disease in humans.14. The nude rodent model for human neoplastic disease of claim 13,wherein said rodent is a rat.
 15. An immunodeficient rodent model forprogression of human neoplastic disease, the progression of said diseasebeing characterized by growth of a primary tumor site and metastasis tosecondary tumor sites, wherein said rodent has histologically intacthuman neoplastic tissue of at least 1 mm³ in size transplanted onto anorgan of said rodent which corresponds to the human organ from whichsaid tissue is originally obtained; and has sufficient immuno-deficiencyto allow said transplanted neoplastic tissue to grow at said primarysite and metastasize to said secondary tumor sites, so as to mimic theprogression of the neoplastic disease including the metastatic behaviorof said neoplastic disease in humans.
 16. The immunodeficient rodentmodel for human neoplastic disease of claim 15, wherein said rodent is arat.
 17. The immunodeficient rodent model for human neoplastic diseaseof claim 15, wherein said rodent is a mouse.
 18. The immunodeficientrodent model for human neoplastic disease of claim 17, wherein saidrodent is a severe combined immunodeficient (SCID) mouse.
 19. A methodof generating a nude rodent model for progression of human neoplasticdisease, the progression of said disease being characterized by growthof a primary tumor site and metastasis to a secondary tumor sites, saidmethod comprising: transplanting histologically intact human neoplastictissue of at least 1 mm ³ in size onto an organ of a nude rodent whichcorresponds to the human organ from which said tissue is originallyobtained; and allowing said transplanted tissue to grow at said primarysite and metastasize to said secondary tumor sites, so as to mimicprogression of the neoplastic disease including the metastatic behaviorof said neoplastic disease in humans.
 20. The method of generating anude rodent model for human neoplastic disease of claim 19, wherein saidrodent is a rat.
 21. A method of generating an immunodeficient rodentmodel for progression of human neoplastic disease, the progression ofsaid disease being characterized by growth of a primary tumor site andmetastasis to secondary tumor sites, said method comprising:transplanting histologically intact human neoplastic tissue of at least1 mm ³ in size onto an organ of an immunodeficient rodent whichcorresponds to the human organ from which said tissue is originallyobtained; and allowing said transplanted tissue to grow at said primarysite and metastasize to said secondary tumor sites, so as to mimicprogression of the neoplastic disease including the metastatic behaviorof said neoplastic disease in humans.
 22. The method of generating animmunodeficient rodent model for human neoplastic disease of claim 21,wherein said rodent is a rat.
 23. The method of generating animmunodeficient rodent model for human neoplastic disease of claim 21,wherein said rodent is a mouse.
 24. The method of generating animmunodeficient rodent model for human neoplastic disease of claim 23,wherein said rodent is a severe combined immunodeficient (SCID) mouse.25. A nude rodent model for progression of human neoplastic disease, theprogression of said disease being characterized by growth of a primarytumor site and metastasis to secondary tumor sites, wherein said rodenthas histologically intact human neoplastic tissue transplanted onto anorgan of said rodent which corresponds to the human organ from whichsaid tissue is originally obtained; and has sufficient immuno-deficiencyto allow said transplanted neoplastic tissue to grow at said primarysite and metastasize to said secondary tumor sites, so as to mimic theprogression of the neoplastic disease including the metastatic behaviorof said neoplastic disease in humans.
 26. An immunodeficient rodentmodel for progression of human neoplastic disease, the progression ofsaid disease being characterized by growth of a primary tumor site andmetastasis to secondary tumor sites, wherein said rodent hashistologically intact human neoplastic tissue transplanted onto an organof said rodent which corresponds to the human organ from which saidtissue is originally obtained; and has sufficient immuno-deficiency toallow said transplanted neoplastic tissue to grow at said primary siteand metastasize to said secondary tumor sites, so as to mimic theprogression of the neoplastic disease including the metastatic behaviorof said neoplastic disease in humans.
 27. A nude rodent model accordingto claim 13 wherein said neoplastic tissue is selected from breasttissue, ovarian tissue or pleural tissue.
 28. A nude rodent modelaccording to claim 27 wherein said neoplastic tissue is obtained fromhuman breast tissue.
 29. A nude rodent model according to claim 27wherein said neoplastic tissue is obtained from human ovarian tissue.30. A nude rodent model according to claim 27 wherein said neoplastictissue is obtained from human pleural tissue.
 31. An immunodeficientrodent model according to claim 15 wherein said neoplastic tissue isselected from breast tissue, ovarian tissue or pleural tissue.
 32. Animmunodeficient rodent model according to claim 31 wherein saidneoplastic tissue is obtained from human breast cancer.
 33. Animmunodeficient rodent model according to claim 31 wherein saidneoplastic tissue is obtained from human ovarian tissue.
 34. Animmunodeficient rodent model according to claim 31 wherein saidneoplastic tissue is obtained from human pleural tissue.
 35. The methodaccording to claim 19 wherein said neoplastic tissue is selected frombreast tissue, ovarian tissue or pleural tissue.
 36. The methodaccording to claim 35 wherein said neoplastic tissue is obtained fromhuman breast tissue.
 37. The method according to claim 35 wherein saidneoplastic tissue is obtained from human ovarian tissue.
 38. The methodaccording to claim 35 wherein said neoplastic tissue is obtained fromhuman pleural tissue.
 39. The method according to claim 21 wherein saidneoplastic tissue is selected from breast tissue, ovarian tissue orpleural tissue.
 40. The method according to claim 39 wherein saidneoplastic tissue is obtained from human breast tissue.
 41. The methodaccording to claim 39 wherein said neoplastic tissue is obtained fromhuman ovarian tissue.
 42. The method according to claim 39 wherein saidneoplastic tissue is obtained from human pleural tissue.
 43. A nuderodent model according to claim 25 wherein said neoplastic tissue isselected from breast tissue, ovarian tissue or pleural tissue.
 44. Anude rodent model according to claim 43 wherein said neoplastic tissueis obtained from human breast tissue.
 45. A nude rodent model accordingto claim 43 wherein said neoplastic tissue is obtained from humanovarian tissue.
 46. A nude rodent model according to claim 43 whereinsaid neoplastic tissue is obtained from human pleural tissue.
 47. Animmunodeficient rodent model according to claim 26 wherein saidneoplastic tissue is selected from breast tissue, ovarian tissue orpleural tissue.
 48. An immunodeficient rodent model according to claim47 wherein said neoplastic tissue is obtained from human breast tissue.49. An immunodeficient rodent model according to claim 47 wherein saidneoplastic tissue is obtained from human ovarian tissue.
 50. Animmunodeficient rodent model according to claim 47 wherein saidneoplastic tissue is obtained from human pleural tissue.